Project Summary The long term goal of this project is to uncover the pathways by which the transcriptional co-activator and oncogene YAP is regulated by the LATS and SRC kinases in response to mechanical stimuli, which could lead to better treatments for cancer, and improved stem cell therapies. The terminal Hippo pathway kinase LATS inhibits YAP nuclear localization, but also has a poorly characterized but important functions in cytokinesis, whereas the SRC tyrosine kinase promotes YAP nuclear localization. YAP nuclear localization is regulated by diverse stimuli such as the actin cytoskeleton, substrate stiffness, cell detachment, cell crowding, and stretch to control density dependent inhibition of growth, tissue repair and stem cell proliferation and differentiation. When in the nucleus, the YAP promotes cell survival and proliferation. Here we will determine how LATS is regulated by both tension at cell-cell junctions to control density dependent inhibition of cell growth and during mitosis to control cytokinesis. Identification of specific mitotic pathways may allow LATS activity to be manipulated to promote tissue regeneration without interfering with cell division. We will also test a hypothesis for how the SRC kinase collaborates with the AMOT protein in response to cell adhesion and other stimuli to target YAP to the nucleus. In Specific Aim 1, we will determine how several proteins that comprise a tension sensor at cell-cell junctions work together to regulate LATS activity in response to cellular tension across sheets of cells. In Specific Aim 2 we will test a hypothesis that SRC turns AMOT from an inhibitor of YAP into an activator. Understanding this pathway will be important for determining whether drugs that increase AMOT levels are appropriate therapies for a given cancer. In Specific Aim 3 we will determine how LATS is activated in mitosis, the identity of its mitotic substrates, and how it promotes cytokinesis. Overall this work will reveal how LATS and YAP are regulated by specific stimuli. These studies will have an important impact on our understanding of tumor suppression and tissue regeneration and may lead to better ways to manipulate these important processes.